Cathepsin S deficiency improves muscle mass loss and dysfunction via the modulation of protein metabolism in mice under pathological stress conditions

Cathepsin S (CTSS) is a widely expressed cysteinyl protease that has garnered attention because of its enzymatic and non‐enzymatic functions under inflammatory and metabolic pathological conditions. Here, we examined whether CTSS participates in stress‐related skeletal muscle mass loss and dysfunction, focusing on protein metabolic imbalance. Eight‐week‐old male wildtype (CTSS+/+) and CTSS‐knockout (CTSS−/−) mice were randomly assigned to non‐stress and variable‐stress groups for 2 weeks, and then processed for morphological and biochemical studies. Compared with non‐stressed mice, stressed CTSS+/+ mice showed significant losses of muscle mass, muscle function, and muscle fiber area. In this setting, the stress‐induced harmful changes in the levels of oxidative stress‐related (gp91phox and p22phox,), inflammation‐related (SDF‐1, CXCR4, IL‐1β, TNF‐α, MCP‐1, ICAM‐1, and VCAM‐1), mitochondrial biogenesis‐related (PPAR‐γ and PGC‐1α) genes and/or proteins and protein metabolism‐related (p‐PI3K, p‐Akt, p‐FoxO3α, MuRF‐1, and MAFbx1) proteins; and these alterations were rectified by CTSS deletion. Metabolomic analysis revealed that stressed CTSS−/− mice exhibited a significant improvement in the levels of glutamine metabolism pathway products. Thus, these findings indicated that CTSS can control chronic stress‐related skeletal muscle atrophy and dysfunction by modulating protein metabolic imbalance, and thus CTSS was suggested to be a promising new therapeutic target for chronic stress‐related muscular diseases.


| INTRODUCTION
It has become clear that chronic psychological stress (CPS) provoked by modern lifestyles is associated with the incidence of various inflammatory and metabolic disorders. Loss of muscle mass is closely linked to both decreased quality of life and poor prognosis of a variety of diseases (e.g., muscle disorders, muscular dystrophy, cachexia, diabetes, and heart failure). 1 These pathological disease states cause skeletal muscle atrophy by imbalanced protein synthesis and degradation in skeletal muscle. 2,3 Skeletal muscle atrophy is often accompanied by an elevation in inflammatory cytokines and oxidative stress production, which induce harmful changes in the metabolism of proteins, carbohydrates, and lipids. 2,4 Emerging evidence suggests that biological oxidative and inflammatory stimulators increase the expression of several proteases, including members of the matrix metalloproteinases and cysteinyl cathepsins (i.e., CTSS and CTSK), which then modulate resident cell events (migration, invasion, apoptosis, and proliferation) and extracellular matrix remodeling in inflammatory and metabolic disorders. 2,5,6,7,8,9 Although it is well-known that chronic stress causes inflammatory actions and oxidative stress production to promote cardiovascular disease initiation and progression in humans and animals under various pathological conditions, [5][6][7] it remains largely uncertain CPS causes skeletal muscle atrophy and dysfunction.
Cathepsins are primary intracellular proteases that act in the recycling of unwanted proteins in lysosomes and protein processing in other intracellular organelles, such as hormone secretory granules. 10,11 Among cathepsins, cathepsin S (CTSS) exhibits potent elastinolytic and collagenolytic activities. Over the past two decades, a number of clinical and laboratory studies have made several important observations that contribute to our understanding of the biological activity of CTSS in human and animal pathobiology. 12 For example, CTSS/transforming growth factor-1β-dependent fibroblast trans-differentiation was shown to contribute to cardiac fibrosis in response to ischemic stress. 13 In addition, CTSS activity was shown to control injury-induced experimental hyperplasia via the modulation of the p38 mitogen-activated protein kinase (p38-MAPK)/protein kinase B (Akt)-histone deacetylases-6 signaling pathway in mice. 14 Limited previous studies reported that CTSK and CTSS were upregulated during various forms of skeletal muscle atrophy. 2,15,16,17 We also later observed that stressed atherosclerotic lesions showed increased expression of CTSS in mice that underwent carotid artery ligation surgery. 5,7 Finally, a single study reported that CTSS induction is a pathologic event that contributes to the pathogenesis of muscular dystrophy in mdx model mice. 17 However, the precise role of CTSS in stress-induced muscle mass loss and dysfunction remains unclear.
The primary aim of the present study was thus to investigate the role(s) of CTSS in the skeletal muscle atrophy and dysfunction in wildtype (CTSS +/+ ) and CTSSknockout (CTSS −/− ) mice under chronic variable stress conditions, with a special focus on the CTSS-mediated modulation of inflammation, oxidative stress, and protein metabolic imbalance. We also performed a proteome array and metabolomic analysis to evaluate changes in plasma inflammatory factors and muscle metabolites. Based on our present results, we propose that CTSS is an important molecular determinant of muscle atrophy in patients with CPS and a potential therapeutic target.

| Animal care and use
The mice used in this study were 7-week-old male CTSS −/− (knockout, KO) and CTSS +/+ (wildtype, WT) C57BL/6J mice weighing 22-24 g. The mice were housed in a temperature-controlled room (22° ± 2°C, 50% ± 5% humidity) with a 12-h light-dark cycle, with ad libitum access to food and water. The animal protocol (Protocol No. 27304) was approved by the Institution Animal Care and Use Committee of Nagoya University and performed according to Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. metabolism pathway products. Thus, these findings indicated that CTSS can control chronic stress-related skeletal muscle atrophy and dysfunction by modulating protein metabolic imbalance, and thus CTSS was suggested to be a promising new therapeutic target for chronic stress-related muscular diseases.

K E Y W O R D S
cathepsin Sinflammationmuscle atrophystress, oxidative stress

| Stress procedure
For the evaluation of effects of chronic stress and the role of CTSS on the metabolic pathways of skeletal muscle, after 1 week of rest, 8-week-old male CTSS +/+ and CTSS −/− mice were randomly assigned to a non-stress group or stress group for 2 weeks. The control mice were left undisturbed and allowed contact with each other. Mice were exposed to chronic variable stress for 2 weeks. For the immobilized stress, the mouse was kept in an animal stress cage (cat. no. 155-BSRR; Natsume, Seiakusho, Tokyo) for 4 h per day (from 9 a.m. to 1 p.m.) 7 days/week without food and water. We administered three different combinations of stressors over each week from Monday to Sunday with the order of stressors within each combination changed randomly to prevent the mice from becoming accustomed to the restraint stress 18 : (1) Horizontal cage and damp: We removed the sawdust from the floor of the stress cage and placed some water in the cage; the stress cage was then suspended horizontally, with the mouse's tail in the water for 4 h once every 2 days; (2) Cage tilt: We put the mouse in the stress cage and suspended the cage at a 45° angle for 4 h once every 2 days; (3) Overnight illumination: The mouse was placed separately in a cage in a room with all-night lighting (from 9 p.m. to 9 a.m.), 3×/week.

| Sample collections
At the end of the stress periods, the mice from all four groups were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Dainippon Pharmaceutical, Osaka, Japan), and both arterial blood samples from the left ventricle and muscle tissue were collected. On stress day 14, both sides' gastrocnemius muscles were rapidly dissected, weighed, and consecutively numbered. For the biological analysis, the right gastrocnemius was isolated and maintained in RNA later solution (for the gene assay) or stored at −80°C (for the protein assay); for the morphological analysis, the left gastrocnemius muscles were rapidly frozen in liquid nitrogen-cooled isopentane, and finally stored at −80°C (CW: CTSS +/+ control mice, n = 8; CK: CTSS −/− control mice, n = 8; SW: 14-day-stressed CTSS +/+ mice, n = 8; SK: 14-day-stressed CTSS −/− mice, n = 8).

| Evaluation of body weight and grip strength
The body weights of all mice were measured on days 0, 7, and 14 after the stress period. At the same time, a blinded forelimb grip strength test was performed once a week using a commercial digital grip strength meter (Columbus, Largo, FL). Mice held by the tail were allowed to grasp a wire grid with the forepaws. The mice were then gently pulled by the tail until they released their grip. The force achieved by the mouse was recorded during the trials and averaged. 2

| Routine blood tests
The whole blood with EDTA was taken using routine blood tests. The routine blood tests were carried out using a Sysmex XN-1000v [B1] automated hematology analyzer (SYSMEX Co., Tokyo).

| Proteome profiler array
A mouse angiogenesis array kit (R&D Systems, Minneapolis, MN) and mouse XL cytokine array kit (R&D Systems) were used according to the manufacturer's instructions. In brief, blood samples were allowed to clot for 2 hrs at room temperature before centrifuging for 15 min at 2000× g. The serum samples were collected and stored at −80°.

| Elisa
A mouse elisa kit (R&D Systems) was used for the analysis of interleukin interferon (IFN)-γ, IL-18 and IL-1β. We used 50 μL of whole blood with EDTA according to the manufacturer's instructions.

| Quantitative real-time gene expression essay
Total RNAs were isolated from gastrocnemius muscle with RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The mRNA was reverse-transcribed to cDNA with a superscript III firststrand synthesis system (Invitrogen, Carlsbad, CA) for a quantitative polymerase chain reaction (qPCR) assay. qPCR was performed using a power SYBR green PCR master mix (Applied Biosystems, Foster City, CA) and appropriate primers (Table 1) in an ABI7300 real-time qPCR system. A conventional polymerase chain reaction (PCR) protocol was also performed for targeted gene expressions, with the following conditions: 95°C for 10 min followed by 45 cycles at 95°C for 30 s and 55°C for 1 min, followed by 1 cycle each at 95°C for 15 s, 60°C for 30 s, and finally 95°C for 15 s. The data are shown relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

| Morphometry analyses
We prepared serial gastrocnemius skeletal muscle crosscryosections (4 μm thick) at a rate of 3-4 sections every 40 μm and stained them with hematoxylin and eosin (H&E). The area per muscle fiber was measured in three randomly chosen microscopic fields from six different sections in each tissue block and averaged for each mouse.

| Metabolomic analysis
Gastrocnemius muscles of mice were used for the metabolomic analysis (Human Metabolome Technologies Inc., Tsuruoka, Japan). 30 mg of the frozen mouse muscle sample was transferred into 500 μL of methanol containing 50 mM of the external standard. After five rounds of homogenization by BMSM10N21 (BMS, Tokyo) at 1500 rpm for 120 s each, 500 μL of chloroform and 200 μL of ultrapure water were added to the homogenate, mixed well, and centrifuged at 2300 g for 5 min at 4°C. Then, 350 μL of the upper aqueous layer was centrifugally filtered through a T A B L E 1 Primer sequences for mice used for quantitative real-time PCR. CTSS  GTGGCCACTA AAGGGCCTG  ACCGC TTT TGT AGA AGA AGA AGGAG   VEGF  CTGCC GTC CGA TTG AGACC  CCCCT CCT TGT ACC ACTGTC   SDF-1  TGAGC GAG TAC AAC AAGGGC  GGCTG GTC ATG GAA AGG ACAG   CXCR4  CTTCT GGG CAG TTG ATG CCAT  CTGTT GGT GGC GTG GACAAT   IL-17  TCAGC GTG TCC AAA CAC TGAG  CGCCA AGG GAG TTA AAG ACTT   IL- Millipore 5-kDa cutoff filter to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 μL of Milli-Q water for a capillary electrophoresis-mass spectrometry (CE-MS) analysis. 19 In brief, CE-MS experiments were performed using an Agilent CE system equipped with a time-of-flight mass spectrometer (TOF-MS) and a builtin diode-array detector (Agilent Technologies, Santa Clara, CA). Cationic metabolites were analyzed using a fusedsilica capillary (50 mm i.d., 680 cm total length) with cation buffer solution (Human Metabolome Technologies) as the electrolyte. The samples were injected at a pressure of 5.0 kPa for 10 s (approximately 10 nL). The applied voltage was set at 30 kV. Electrospray ionization mass spectrometry (ESI-MS) was conducted r54in the positive ion mode, and the capillary voltage was set at 4000 V. The spectrometer was scanned from m/z 50 to 1000. Other conditions were the same as in the cation analysis (Soga & Heiger 20 ). Anionic metabolites were analyzed using a fused-silica capillary (50 mm i.d., 680 cm total length), with anion buffer solution (Human Metabolome Technologies) as the electrolyte. The samples were injected at a pressure of 5.0 kPa for 25 s (approximately 25 nL). The applied voltage was set at 30 kV. ESIMS was conducted in the negative ion mode, and the capillary voltage was set at 3500 V. The sample in the spectrometer was scanned from m/z 50 to 1000. Other conditions were the same as described for the anion analysis. Metabolites in the samples were identified by comparing the migration time and m/z ratio with authentic standards, and differences of 60.5 min and 610 ppm, respectively, were permitted. The identified metabolites were quantified by comparing their peak areas with those of authentic standards using ChemStation software (Agilent Technologies). The samples obtained were then subjected to capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) analysis using the Agilent CE-TOFMS system (Agilent Technologies) at 4°C. The detected peaks were aligned according to their m/z values and normalized migration times. The peaks were mean-centered and scaled using their standard deviations on a per-peak basis as a pretreatment. After applying autoscaling, a principal component analysis (PCA) and a hierarchical clustering analysis (HCA) were conducted using JMP ver. 11 software (SAS Institute, Cary, NC). In the PCA, a score plot of the first and second principal components was generated. In the HCA, the resulting data sets from each genotype were clustered by Euclidean distance using Ward's method. 21 Heat maps were generated by coloring the values of all data across their value ranges. 19

| Mitochondrial function study
According to the Na + -K + -ATPsae activity kit protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the gastrocnemius muscle tissue was weighed and normal saline was used for dilution, then measured the release of inorganic phosphate from ATP with malachite green dye method to access the activity of Na + -K + -ATPsae. The mitochondrial complex IV activity assay kit (Solarbio, Beijing, China) was used to measure the activities of mitochondrial complex to IV. In brief, the gastrocnemius muscle tissue was weighed 0.1 g, and the analysis was performed following the manufacturer's instructions.

| Statistical analysis
Data are expressed as mean ± SEM. Student's t-tests were used for comparisons between the two groups. Body weight and grip strength data were subjected to two-way repeated-measures ANOVA and Bonferroni post hoc tests. Origin software ver. 6 1 was used, and values of p < .05 were considered statistically significant. All parameter calculations were conducted by two observers blinded to the treatment of the mice.

| RESULTS
For the investigation of the impacts of chronic stress on the expression of CTSS and muscle disorder, 8-week-old mice were subjected to a 2-week variable stress protocol ( Figure 1A). As shown in Figure 1B, we observed that there was no difference between the 14-day-stressed CTSS +/+ (SW) mice and the 14-day-stressed CTSS −/− (SK) mice. In contrast, the body weight of SW mice was significantly lower than those of SK mice ( Figure 1C).

| CTSS deletion alleviates stress-induced increases in inflammatory biomarkers in the blood
Next, we examined the peripheral blood of the mice to observe the change in inflammatory cells. Blood routine examination showed an increase in peripheral white blood cells, lymphocytes, mononuclear cells and neutrophils after stress, and CTSS deficiency mitigated these changes ( Table 2). A proteome array was performed using the serum of mice (Figure 2A-D) and ELISA was performed using plasma ( Figure 2E). We observed that stress resulted in increases in the levels of SDF-1, endothelin-1, IGFBP-2, IGFBP3, IL1ɑ, osteopontin, angiopoietin, CCL11, CCL21, and complement factor 21 proteins and that CTSS −/− reversed these protein changes after stress (Figure 2A-D). The results of ELISA showed that the levels of IL-18, IL-1β, and IFN-γ increased after stress, and these changes were rectified by CTSS deletion (Figure 2E).

| CTSS deficiency can reverse stress-induced increases in inflammation in gastrocnemius muscle
Since we found significant changes in SDF-1 levels in the blood, we next investigated the changes of SDF-1 in gastrocnemius muscle at the protein and mRNA levels. As anticipated, we observed that stress increased the levels of SDF-1 and CXCR4 protein and mRNA ( Figure 3A-C). As shown in Figure 3A, the qPCR revealed that the stress resulted in an increase in the levels of oxidative stress-related (p22 phox , p67 phox , p47 phox , and gp91 phox ) and inflammation-related (IL-17, IL18, MCP-1, ICAM-1, VCAM-1, and TNF-α) mRNAs, and a decrease in the levels of mitochondrial biogenesis-related genes (PPAR-γ and PGC-1α), and CTSS −/− exerted beneficial effects on the levels of the investigated genes. In addition, stress resulted in a reduction in the levels of the Na + -K + -ATPase and mitochondrial complex IV activities of the gastrocnemius muscle tissues; and these changes were rectified by the CTSS deletion ( Figure 3D,E). These results suggest that stress-induced skeletal muscle change might be closely linked to the oxidative stress production, inflammation, and mitochondrial damage.

| CTSS gene knockout regulates muscle atrophy through the SDF-1/CXCR4-p-PI3K/p-Akt signaling pathway
Gastrocnemius muscle weight and grip strength ( Figure 4A,B) were markedly reduced in CTSS +/+ stressinduced (SW) mice compared to the non-stressed CTSS −/− mice. The grip strength monitoring indicated that the grip strength declined in parallel with the muscle weight in the 14-day-stressed CTSS +/+ mice; these changes were rectified by CTSS −/− . The quantitative data obtained by H&E staining analysis showed that the gastrocnemius muscle-fiber cross-section area was reduced after stress, and the gastrocnemius muscle-fiber cross-section area was higher in the 14-day-stressed CTSS −/− mice than the 14-day-stressed CTSS +/+ mice ( Figure 4C-E). Therefore, we also observed that stress resulted in decreases in the levels of p-PI3K/ p-Akt proteins located downstream of the SDF-1/ CXCR4 signaling pathway and that CTSS −/− reversed these molecular-level changes after stress ( Figure 5A,B). Akt reduces muscle atrophy by phosphorylating FoxO transcription factor to reduce the expression of ubiquitin-related genes. 22 As shown in Figure 5A-C, western blot analysis revealed that stress also caused a change in the levels of p-FoxO3ɑ, MuRF-1, and MAFbx1 proteins, and these changes were rectified by CTSS deletion. These observations thus indicate that CTSS might act as a key mediator of the harmful oxidative stress, inflammation, and apoptosis that occur in mice in response to chronic stress injury.

| Metabolomic analysis of gastrocnemius muscle
In the gastrocnemius muscle metabolomic analysis, 192 peaks were detected by the cation and anion modes of CE-MS. The first principal component effectively and clearly distinguished CW from the other three groups (X axis). As shown in Figure 6A, the results of PCA of the measured peaks suggested that stress-induced changes in gastrocnemius muscle resulted in significant changes in the metabolite. As demonstrated by the heat map analysis ( Figure 6B), CW was significantly different from the other three groups. Taken together, the PCA and heat map results support the idea that stress significantly affects the metabolite profile of skeletal muscle, leading to a clear separation from the other three groups, but CTSS deletion can prevent this change.
As shown in Figure 7, we analyzed the glutamine metabolism pathways, which metabolizes glutamine, glutamic acid and GABA, and the choline metabolism pathway, which is responsible for the metabolism of choline, pangamic acid, sarcosine, glycine, and serine. All of these parameters changed significantly after stress in WS mice, while the SK mice exhibited a marked improvement in these parameters.

| DISCUSSION
This study demonstrated that chronic stress-induced losses of skeletal muscle mass and function were associated with inflammation, oxidative stress, and protein metabolism imbalance. The significant finding of this study is that mice lacking the CTSS gene were resistant to chronic stress-induced muscular atrophy. At the molecular level, CTSS −/− was found to prevent (i) Akt/mTOR-dependent anabolic signaling inactivation; (ii) FoxO3a-MuRF-1/ MAFbx1-dependent catabolic pathway activation; (iii) TNF-α/SDF-1-CXCR4-mediated inflammation and NADPH oxidase-mediated oxidative stress production; (iv) glutamine and choline metabolism pathway activation. To the best of our knowledge, this is the first study to report that CTSS deficiency-mediated muscle-protective actions might be at least partly attributable to the inflammation-and oxidative stress-mediated Akt/mTOR inactivation and FoxO3a-MuRF-1/MAFbx1 activation, which led to the prevention of protein turnover in mice under our experimental conditions (Figure 8).
Chronic stress has been shown to enhance the inflammatory response in different tissues (e.g., adipose and vascular tissues). 2,23 Systemic illnesses can trigger muscle atrophy by elevating proinflammatory/catabolic cytokines (TNF-ɑ, IL-1ɑ, IL-1β). 24 A previous metaanalysis using correlation data found that independent of the disease state, higher levels of C reactive protein, IL-6, TNF-ɑ, and other systemic inflammatory markers appeared to be associated with lower muscle strength and muscle mass. 25 In this study, we found that stress causes an increase in white blood cells, and increases in the  important mediator of stress-related muscle disorder in mice.
A recent comprehensive review documented the close relationship between reactive oxygen species and skeletal muscle diseases. 27 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the key sources of reactive oxygen species in immune cells. 28 The phagocyte NADPH oxidase is composed of two membrane subunits, gp91 phox and p22 phox , three cytosolic subunits, p40 phox , p47 phox , and p67 phox , and the small GTPase Rac. Our present work showed that CTSS −/− ameliorated the elevated NADPH oxidase subunit expressions (p22 phox , p47 phox , p67 phox , and gp91 phox ). Recently, we demonstrated that chronic stress accelerated kidney injury and remodeling via the modulation of oxidative stress-induced CTSK expression and activity in mice that received kidney 5/6 nephrectomy surgery. 6 Collectively, these observations suggest that increased oxidative stress-mediated CTSS overexpression could also contribute to the mechanisms underlying stress-related skeletal muscle damage and atrophy in mice.
SDF-1 is constitutively expressed in multiple organs, including the skeletal muscle, heart, kidney, skin, and bone marrow. 29 SDF-1 secretion is also associated with tissue damages such as heart infarct, limb ischemia, excessive bleeding, total body irradiation, and chemotherapy-related tissue damage. 30 CXCR4 is the main specific receptor for SDF-1 and amplifies the signaling of SDF-1. The SDF-1/ CXCR4 signaling cascade has a regulatory effect on the expression of cytokines and chemokines. SDF-1 has been shown to upregulate TNF-ɑ mRNA and protein secretion, as well as TNF-receptor 2 expression. 31 Our present results showed that the levels of expression of SDF-1/CXCR4 genes and proteins are increased in the stressed skeletal muscle, and these changes were reversed by CTSS −/− , suggesting that CTSS may act as a regulator of SDF-1/CXCR4mediated inflammatory responses in mice subjected to chronic stress.
CTSS and CTSK genes were increased in damaged skeletal muscles. 15 The signaling pathway that controls muscle ubiquitin ligase activation begins with a reduction in the activity of the PI3K/Akt pathway, which leads to the activation of FoxO transcription factors and induction of MAFbx1, followed by effects on the MAFbx1 promoter by the persistently activated FoxO3, finally causing transcription of MAFbx1 and significant atrophy of myotubes and myofibers. 32 It was reported that the ubiquitin-proteasome pathway promotes protein degradation through protein polyubiquitination. 33 In the ubiquitin-proteasome system, proteins are degraded by the 26S proteasome through the covalent linkage of ubiquitin molecular chains. The rate-limiting step of ubiquitin ligase E3 binds protein substrates and catalyzes the movement of ubiquitin from the E2 enzyme to the substrate, which can affect subsequent proteasome-dependent degradation. 34 The musclespecific E3 ubiquitin ligase atrophy gene 1/MAFbx1 and MuRF-1 play a role in the process of muscle atrophy. 35,36 In our present experiments, we observed that CTSS was upregulated during stress-induced muscle atrophy. And the CTSS-lacking mice were resistant to stress-induced morphological skeletal muscle myofiber shiner and mass loss and a functional decline in muscle strength. In this setting, we observed that CTSS −/− resulted in increased levels of p-PI3K, p-Akt, p-FoxO3α and decreased levels of MAFbx1 and MuRF-1 in the stressed skeletal muscles. Thus, an imbalance between CTSS activation-mediated PI3K/Akt-FoxO3α and MAFbx1-MuRF-1 signaling might be a common mechanism in the skeletal muscle atrophy and dysfunction in mice under stress conditions. In contrast, CTSS deletion prevented a reduction in the Na + -K + -ATPase and mitochondrial complex IV activities of the stressed gastrocnemius muscle tissues, suggesting that the increased CTSK expression-related mitochondrial dysfunction might also contribute to muscle injury and dysfunction in mice under our experimental conditions.
The results of metabolic analysis revealed that CTSS deletion partially reversed the reduction of the glutamate/ GABA (gamma-aminobutyric acid)-glutamine, serine/ glycine, betaine, and sarcosine levels in gastrocnemius muscle tissues. Glutamate is mainly synthesized in skeletal muscle and has many regulatory functions, including regulation of the rate of protein synthesis and reduction of the rate of protein degradation, whereas its intramuscular deficiency may directly contribute to lean body mass loss 37 This could also explain the weight loss of stressed mice in other ways. That is, GABA can be directly synthesized by decarboxylation of glutamic acid, and the glutamic acid/ GABA-glutamine metabolic cycle plays an important role in maintaining the balance of amino acid neurotransmitters. 38 Additionally, glycine is one of the three amino acids that comprise the tripeptide glutathione (glycine, glutamate, and cysteine), and serine can be metabolized to cysteine, the serine/glycine/glutamic acid restriction reduced glutathione levels, glutathione, as a major intracellular antioxidant, increases reactive oxygen species production. 39 When serine/glycine is decreased, reactive oxygen species production may be regulated beyond normal levels in a compensatory manner in response to cell cycle arrest. 40 On the contrary, bataine is a methyl derivative of glycine first isolated from the sugar beet, and it has been shown to exert the most influential effects on muscle growth under conditions of metabolic or nutritional stress. 41 Sarcosine is derived from glycine and arginine, which has a different role in the energy transduction in different tissues that contain 95% of the total creatine pool in skeletal muscle. Sarcosine administration improves muscle utilization by increasing the concentration of creatine phosphate, and improving the rephosphorylation of adenosine diphosphate to adenosine triphosphate. 20 CTSS deficiency prevents the suppression of glutamine and choline metabolism pathways in mice under stress conditions. Taken together, these data suggest that the musculoprotective actions are mediated, at least in part, through amelioration of the impaired glutamine and choline metabolism pathways in CTSS −/− mice under chronic stress conditions. Study limitations should be recognized. First, the chronic variable stress model used herein is an animal stress model that cannot completely mimic human psychological stress. Second, our in vivo study was not combined with in vitro experiments to clarify the exact mechanisms of CTSS −/− -mediated muscle benefits under our experimental conditions. Lastly, we did not evaluate the impact of chronic stress on mobility in both genetic mice. Further research is necessary to investigate these issues.

| CONCLUSIONS
In summary, the expressions of the CTSS gene and protein were increased in the skeletal muscle tissues of mice subjected to chronic stress. CTSS deletion alleviated the stress-induced loss in chronic stress-related skeletal muscle mass and skeletal muscle function in mice, suggesting that selective CTSS inhibition might have potential utility in the management of stress-related muscle disorders.